Photosynthesis Research 30: 49-59, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.
Historical corner
The legacy of Hans Molisch (1856-1937), photosynthesis savant Howard Gest
Photosynthetic Bacteria Group, Biology Department and Department of History and Philosophy of Science, Indiana University, Bloomington, IN 47405, USA Received 19 August 1991; accepted 29 August 1991 'It is a noble employment to rescue from oblivion those who deserve to be remembered'; Pliny, the Younger, Letters, V, no VIII; to Titinius Capito.
Abstract
Hans Molisch (1856-1937) was an exceptionally gifted and productive researcher who had broad interests in plant biology, physiology and biochemistry. In addition, he pioneered in isolating a number of species of purple photosynthetic bacteria in pure culture (including Rhodobacter capsulatus), which facilitated his discovery of basic aspects of bacterial photosynthesis. Molisch demonstrated conclusively that molecular oxygen is not produced by photosynthetic bacteria, and discovered the photoheterotrophic growth mode. The range of Molisch's research accomplishments was impressive, and he emerges as a major figure in the history of photosynthesis research. This essay reviews the numerous research contributions made by Molisch, particularly in regard to advancing knowledge of the several forms of photosynthetic metabolism. An English translation of his 1914 paper on the photosynthetic creation of visual images on leaves is included as an Appendix.
Hans Molisch (Fig. 1) was born December 6, 1856 in Brtinn, Moravia, now called Brno, Czechoslovakia. When he was nine years old, he had a scientific encounter that was to remain a vivid memory for the rest of his life. During Molisch's youth, Br/inn was a city with a strong commitment to the natural sciences, and Molisch describes the memorable incident in his autobiography (Molisch 1934): 'As a nine-year-old boy I had an interesting experience at a grape harvest festival. One of the (Molisch's) vineyards, called the "King", lay just behind the walls of the monastery of the same name and the prelate Gregor Mendel was the head of the Augustinian chapter there. Earlier, as a secular priest, he was professor of natural sciences at the high school (Br/inn Technical Secondary School) in the Johannesgasse where my two brothers, Ferdinand and Edmund, had the good fortune of being his pupils. Mendel was very favorably disposed to our family. When
Ferdinant was in Dresden for a year at the well-known Wagner nursery in order to learn especially about azealeas, rhododendron and heather, the abbot visited him while passing through, which one can fully appreciate today, after Mendel has become famous as a biologist. On one of the days of the grape harvest, the prelate came over to us from the neighboring monastery and greeted my mother, who was pleasantly surprised by this important visit. I still remember that charming man, of medium height, wearing the priest's black cassock, highly polished boots, gold-rimmed glasses, and engaging features. As my mother presented him the sweetest of grapes on a tray, he spoke to me and my sister, at times seriously, at times joking in an affable manner. At the time, none of us had any inkling that he would achieve world fame in science equal to that of Darwin.' This encounter occurred in 1865, the same year in which Mendel published his findings on heredity.
50 at the University of Sendai in Japan. He accepted, remained for four years, and published his numerous observations in a book entitled Pflanzenbiologie in Japan (Molisch 1926). One of the numerous experimental investigations described concerned the microbiological flora of hot springs. His observations on the growth temperature limits of cyanobacteria, green algae, and typical purple bacteria were confirmed by later investigators. Upon his return to Vienna, Molisch served as Rector of the University for two years and in 'retirement' continued productive research and writing until the end of his life. His outstanding accomplishments were recognized by numerous honorific degrees and appointments (H/After 1939a,b).
Bioluminescence
Fig. 1. Hans Molisch (1856-1937), from an obituary by Kiister (1937).
Molisch's academic career
The details of Molisch's early education have been summarized in obituaries (Hrfler 1939a,b) written by Karl H/After-one of his students in Vienna. These list all of Molisch's publications, some 273, including 20 books. His extraordinary productivity was all the more remarkable considering the breadth of his interests and expertise. He was a plant scientist in the broadest sense, but also an excellent chemist and accomplished microbiologist. Molisch held three professorial appointments before his formal 'retirement' in 1922: 1889-1894, Professor in the Technische Hochschule, Graz; 1894-1908, Professor in the German University, Prague; 1908-1922, Professor of Botany and Director of the Plant Physiology Institute, University of Vienna. In 1921, Molisch was invited to do research, teach, and establish a modern biological institute
Molisch had the reputation of being an outstanding academic teacher who inspired enthusiasm in his students. One of his special interests was bioluminescent bacteria, which he isolated in pure culture from various sources: 'He made a living "bacterial lamp" using Pseudomonas lucifera which burned brightly emitting cold light that could be seen at a distance of 60 paces. Every year Molisch devoted one session of his lecture course to plant luminescence. As the classroom was darkened, one began to see the most wondrous phenomena.., luminescent fish and luminescent eggs (luminescence due to bacterial contaminants) in glass containers. Then there were meter-long tubes filled with a bouillon of luminescent bacteria which appeared dark at first, but which lit up like rockets when Molisch turned them upside down to let an air bubble rise in t h e m - the bacteria would light up only in the presence of oxygen. There were also photographs taken with the light produced by luminescent bacteria, and seedlings which bent towards the luminescence produced in test tubes. Among all these wondrous phenomena, Molisch appeared like a sorcerer' (H/After 1939a). Molisch's research with bioluminescent microorganisms, which were then classified as plants, were described in his book Leuchtende Pflanzen (Molisch 1904).
51
Iron nutrition; pigments Iron Molisch's monograph Die Pflanze in ihren Beziehungen zum Eisen (Molisch 1892) summarized his studies on the various forms of iron in higher plants, algae, fungi and bacteria. One chapter is entitled 'Does chlorophyll contain iron?' Iron was known to be involved in chlorophyll synthesis in some unknown way, and Molisch approached the problem of the role of iron by analyzing purified chlorophyll preparations for the metal. He established clearly that iron was not a constituent of the pigment itself, and attributed traces of iron in the best preparations to contamination. Molisch calculated that if the iron really were an integral component, the formula of the chlorophyll molecule would indicate such a collosal size that no chemist would consider it possible on theoretical grounds, and gave as an example the formula C37300H74600
037300" A continuing interest in the biological roles of iron led to Molisch's classic monograph Die Eisenbakterien (Molisch 1910). His experimental results contradicted some interpretations of Winogradsky on the physiology of iron bacteria, and were summarized in Marjory Stephenson's influential book Bacterial Metabolism (Stephenson 1949) as follows: 'One of the principal opponents of Winogradsky's views was Molisch, who himself made important contributions to the study of the group. He was the first to obtain Leptothrix ochracea in pure culture, and showed that it is not a strict autotrophant, but can grow in peptone, with or without iron. Furthermore, he showed that if in such media manganese replaces iron, the oxide of the former metal is deposited in the sheath of the organism. From these observations he concluded that iron plays no essential part in the metabolism of this organism, and that the deposit of oxides of iron (or manganese) in the sheath is due to adsorptive processes and has no connection with any metabolic function, being, in fact, paralleled by iron accretions in certain algae, moulds, infusoria, and flagellates where no physiological role is assigned to it.' Molisch's conclusions were correct in that to this day, no one has been able to
unambiguously demonstrate that Leptotrix ochracea can grow autotrophically with energy provided by iron oxidation (Brock and Madigan 1991). Pigments Pigments of photosynthetic organisms were also of special interest to Molisch, who made notable research contributions in this connection. In addition to studies on chlorophyll, he pioneered in crystallizing and determining general characteristics of phycoerythrin (Molisch 1894), phycocyanin (Molisch 1895) and anthocyanin (Molisch 1905). An early treatise on anthocyanin pigments (Wheldale 1916) discusses Molisch's various contributions and summarizes his 1905 paper as follows: 'Important account of a number of cases of crystalline and amorphous anthocyanin in tissues of living plants. Also of preparation of crystalline anthocyanin outside the cell. Summary of chief researches and other work on anthocyanin.' With the purple bacteria (Molisch 1907), he demonstrated that bacteriochlorophyll ('bacteriochlorin') was responsible for the pigment absorbancies in the infra-red and at 590 nm, whereas the major absorbancies at lower wavelengths were due to carotenoids ('bacteriopurpurin'), which he succeeded in crystallizing.
Starch pictures Molisch's important work on purple photosynthetic bacteria, described in more detail later in this article, was not in the mainstream of his major efforts, which were primarily directed to plant biology, physiology, and biochemistry. He was an expert in plant anatomy and microchemistry, and in 1886 he developed a very sensitive color reaction for detection of carbohydrates which became widely known as the 'Molisch Reaction.' In my undergraduate college course in biochemistry, 50 years ago, I first heard the name Molisch in this connection. Molisch's interest in carbohydrates led him to devise the so-called 'starch picture' (Molisch 1914, 1920), a clever and dramatic demonstration of green plant photosynthesis. Figure 2 shows a 'starch picture' of Jan Ingen-Housz, the
52 In order to reveal the pattern of starch granules produced photosynthetically while Ingen-Housz's image was projected, the leaf was 'developed' after extraction of the chlorophyll and other leaf pigments. To accomplish the latter, the leaf was dropped into a beaker containing boiling 80% alcohol. The blanched leaf was then placed in a Petri dish and flooded with an iodine-Kl solution to stain the starch granules. Within minutes, the image of Ingen-Housz dramatically appeared on the leaf surface. Thus, the leaf performs as if it were a piece of photographic film, and the 'picture' shows clearly that starch formation is proportional to the light intensity emerging from each point of the negative. Legibility of the inscription at the bottom, which refers to Dr Ingen-Housz's fame as a 'smallpox inoculator' (Gest 1988), attests to the remarkable resolution of the 'photosynthetic photograph.' Molisch (1914) noted that 'in a comparison of the photographic plate with the leaf, the chlorophyll apparatus is related to the lightsensitive silver salt, the starch granule to the silver granule, and the starch iodide reaction to the developer.' A translation of Molisch's original paper on starch pictures is appended. Fig. 2. Starch picture of Jan Ingen-Housz on a geranium leaf, made by Molisch's method (see text), but simplified as described by Walker (1979).
Die Purpurbakterien (1907)
discoverer of photosynthesis (Gest 1988), made using a simplified version of Molisch's procedure. The image was created by the process of photosynthesis, and revealed by staining starch granules as follows. A geranium plant was placed in darkness for two days, to deplete the starch reserves. A leaf with its attached petiole was removed from the plant, and placed on a piece of black cloth that was previously moistened with a dilute solution of baking soda (to provide some CO 2 for photosynthesis). The leaf with its cloth backing was next sandwiched between two plates of glass, and the 'sandwich' fixed in a vertical position with the protruding petiole immersed in a beaker of water. At this point in the procedure, a negative of an engraving of Jan IngenHousz (from Reed 1949) was placed in a slide projector, and Ingen-Housz's image focused on the leaf for about one hour.
Molisch's important contributions to our knowledge of the anoxyphototrophs were described and summarized in his classic monograph (Molisch 1907). He proposed that the various species of purple bacteria should be grouped into two families: Thiorhodaceae, purple sulfur bacteria, and Athiorhodaceae, the non-sulfur purples. Although the families are no longer recognized as such, Molisch's separation of the two physiological types brought significant order to the classification of photosynthetic bacteria, and is still a meaningful and very useful separation. On the basis of numerous experiments, he established that the non-sulfur purple bacteria are widely distributed in nature, and also demonstrated that many types could be readily enriched by providing sources of organic substances, light, 'and restricted oxygen supply.' Molisch isolated and characterized pure cultures of a number of anoxyphototrophs, and
53 many are recognized as type species. These include: Rhodobacter capsulatus, RhodospiriUum photometricum and Rhodopseudomonas palustris. The type species Rhodospirillum rubrum is designated as 'Esmarch/Molisch' because Molisch demonstrated that the organism Spirillum rubrum, isolated as an ordinary heterotroph by Esmarch (1887) from the dried residue of a dead mouse, was in fact a photosynthetic purple bacterium. Another Molisch type species is Amoebobacter (Rhodothece) pendens, a gasvacuolated purple sulfur bacterium he isolated in 1906 (Molisch 1906). van Niel's important 1944 study on purple bacteria (van Niel 1944) leaned heavily on Molisch's observations, and van Niel found little to criticize in this connection. Indeed, Molisch had made notable advances in understanding major biological and physiological features of the anoxyphototrophs. Some of these are reflected by the following excerpts from Die Purpurbak-
terien. 'For the present I am satisfied that the nutritional experiments with purple bacteria have familiarized us with a new type of photosynthesis in which organic substance is assimilated in the light, and whereby the two pigments, the bacteriochlorin and bacteriopurpurin, probably play an analogous role to that of chlorophyll and carotenoid in the carbonic assimilation of green cells.' 'The purple bacteria represent a physiological group of bacteria which, in contrast to other colorless and colored schizomycetes, demonstrate exceptional adjustment to light. The earlier chapters have already given us much information about this. Particularly striking is the influence of light on their movement, already established by Engelmann. In the dark, the purple bacteria sooner or later cease movement; in light, their movements are reactivated. I can essentially confirm what Engelmann says about this in his treatise, rich in physiologically interesting facts.' 'From a phylogenetic standpoint it seems to me that the purple bacteria represent an intermediate stage between colorless bacteria, which efficiently process organic substance without the assistance of light, and the green organisms (i.e., oxygenic) which, in regard to assimilation, adapt
completely to light and have made themselves completely independent of nourishment with organic substance. The purple bacteria are similar to most common bacteria in that they can, to be sure, assimilate organic substances in the dark, but they are also similar to the green organisms insofar as they have adapted themselves to light and have learned to process organic substances in a substantial and better way with the help of light.'
Molisch confirms Engelmann's 'bacteriospectrogramm' Engelmann (1883) established the important fact that purple bacteria respond to infra-red light, as well as to light in the visible portion of the spectrum. At the time, this must have seemed a most surprising finding. Using Bacterium photometricum (a mixture of Chromatium species), and a microspectralocular, Engelmann observed accumulation of the motile cells at specific wavelengths of the spectrum, including a 'band' in the infra-red. The resulting 'bacteriospectrogramm' was very striking (see Fig. 3). In Engelmann's words (Engelmann 1883): 'It requires only a glance at our figure to see that the photokinetically active wavelengths of the visible spectrum are exactly those that are most strongly absorbed; the least effective, those that are absorbed least. The local accumulations of bacteria (Figs. 4 and 5 . . . . not shown here) immediately give the impression of copies of the absorption bands (Figs. 2 and 3 . . . . not shown here). There proves to be a completely analogous relation between absorption and physiological light effect, as has been demonstrated in plant assimilation using the bacterial method. In view of the correspondence, which apparently exists everywhere, between absorption and photokinetics in the visible part of the spectrum, one must suppose that a similar parallelism will exist in the invisible part, especially in the infrared. Accordingly, the wavelengths between about 0.80 and 0.90/.t must also be absorbed very strongly. The only means to validate this are offered by the bacteria themselves.., since the eyes fail to work, and thermoelectric piles and photographic plates are probably not appli-
54
Fig. 3. Photograph of a 'bacteriospectrogramm' showing accumulation of Thiospirillum jenense cells at wavelengths of the spectrum that correspond to photopigment absorbancies. Note in particular the cell accumulation in the infra-red region of the spectrum (bacteriochlorophyll in intact cells of T. jenense absorbs infra-red radiation strongly between ca. 800 and 900 nm). This photograph was made from a frame of a motion picture illustrating the motility and phototactic behavior of the organism. The film was produced in 1968 by Prof Dr Norbert Pfennig at the Institut ffir den Wissenschaftlichen Film, G6ttingen.
cable. One should test to see if infra-red radiation is appreciably attenuated in its effect by passage through the bacteria themselves, as is the case with assimilatory-active light rays upon passage through chlorophyll preparations. Unfortunately, I have as yet been unable to do this experiment (the execution of which does not present real difficulties) because the material was exhausted. Because of the extraordinary sensitivity of our bacteria to infra-red radiations, one must work with dim light and as far as possible with thick absorbing layers. As shown earlier, the latter can be produced with strong continuous local illumination.' Molisch, using pure cultures, confirmed the essential findings of Engelmann, but remarked that the bacteriospectrogramms with certain species were not always as sharp as Engelmann's.
Tests for production of molecular oxygen by purple bacteria The one major difference in the experimental results of Engelmann and Molisch concerned the question: 'Do the purple bacteria produce 0 2
when illuminated?' (Molisch 1907; Ch. III.A.4.). This important aspect was investigated by Engelmann in 1883, who reported negative results. This indicated to Engelmann that the bacterial pigments involved in phototactic behavior of the cells must be fundamentally different from chlorophyll, and that the activating effect of light could not be based on light-dependent assimilation of carbon dioxide, as it is in green plants. Failure to observe 0 2 production apparently was disturbing to Engelmann, and he looked into the question again in 1888 (Engelmann 1888). In the new experiments, he claimed that the bacteria did produce oxygen when illuminated; and thus 'Bacteriopurpurin is a true chlorophyll.' In his monograph, Molisch notes that 'Winogradsky speaks of Engelmann's 1888 results in the following way: 'Without dismissing these new results (since I, for my part, have made no direct experiments on the question), I believe that I can maintain with certainty that the appearances which either do or do not speak in favor of oxygen release in these organisms must have been very inconclusive if Engelmann could obtain directly opposed results with the same organisms at different times according to one and the same method.' The assumption that 0 2 must be, or potentially could be, produced in all photosynthetic processes proved to have a profound effect on experimental design and theories of photosynthesis for a surprisingly long time. In retrospect, it is hard to avoid the conclusion that this mindset had an inhibitory effect on conception of alternative possibilities.
Molecular oxygen in bacterial photosynthesis The concept that molecular oxygen is required in the metabolism of all organisms was already a firm assumption when Louis Pasteur rediscovered (see Bulloch 1938) the existence of fermentative anaerobic bacteria in 1861. The metabolism of anaerobes was interpreted by Pasteur to be based on the capacity of such organisms to remove oxygen gas from organic compounds, such as sugars. Pasteur's explanation (see Fruton 1972) was that the anaerobes were 'similar in all respects to the organisms of the first class (i.e., aerobes), living as they do, assimilating carbon,
55 nitrogen, and phosphates in the same way, and like them requiring oxygen, but differing in that they can, in the absence of free oxygen gas, respire with oxygen gas removed from relatively unstable compounds.' As late as 1884, the influential physiological chemist Felix HoppeSeyler believed that the idea of 'life without air' was improbable, and it must be remembered that it was against this background that Engelmann interpreted and published his observations on Bacterium photometricum. The ambiguity left behind by Engelmann in respect to 0 2 production in bacterial photosynthesis was settled by Molisch in 1907. Although some species of purple bacteria that he examined grew well in the presence of air, most were unable to do so. Between the aerobic and anaerobic extremes, a number of species showed intermediate sensitivities. Molisch concluded that in general, most of the purples tended toward the microaerophilic end of the 'oxygen spectrum.' Using species very sensitive to oxygen, and very sensitive 0 2 detection methods, he was able to make critical tests for photosynthetic oxygen production. The results were invariably negative. Molisch pointed out that if O2-sensitive purple bacteria produced oxygen, as claimed by Engelmann, illumination should cause cells in a suspension to scatter and try to escape from each other, but this does not occur. Thus (Molisch 1907): 'If we illuminate a bacterial field which is so dense that it appears as dark red to the eye, the bacteria nevertheless remain together, and if we lure them into a light trap, then they are drawn by the millions onto a point of light and piled up like the grains on a sandpile. If the rhodobacteria were to release oxygen in the light, such accumulations in light would not be understandable. Hence, I saw in the aforementioned behavior of the purple bacteria new support for my view explicated on pages 41-48 that rhodobacteria in the light are not able to release oxygen.'
tion that 0 2 evolution must/should be an inherent aspect of all photosynthetic processes led Johannes Buder in 1919/1920 (see refs. in van Niel 1941) to propose that purple bacteria evolve oxygen photosynthetically, and simultaneously use the 0 2 for respiratory oxidation of organic compounds or H2S. According to van Niel (1941), Buder 'integrated the controversial ideas of Engelmann, Winogradsky, and Molisch.' During the 1930s there were sporadic reports of 0 2 production by purple bacteria, but studies by van Niel in 1936 (see van Niel 1941) showed that these claims could be explained in other ways. Thus, it is curious that van Niel apparently considered Buder's hypothesis to be a meaningful 'integration' since it was seriously flawed by invoking 0 2 production in bacterial photosynthesis. It is astonishing to me that the notion of possible production of 0 2 by purple bacteria lingered on until 1954 when an investigation using 180 as a tracer confirmed once again that 0 2 is not produced by Rhodospirillum rubrum (Johnston and Brown 1954). In sum, the latent desire of a number of investigators to show that all photosynthetic processes fit a 'unitary pattern' led to futile experimental attempts to demonstrate 0 2 production by purple bacteria over a span of about 70 years! We cannot know the extent to which Engelmann's conclusions in 1888 on 0 2 production by purple bacteria were influenced by prevailing ideas on the 'essential character' of photosynthesis. In any event, his error does not really detract from the great advances made by this versatile and internationally acclaimed scientist whose major field was muscle physiology, with special emphasis on cardiology. Like Molisch, Engelmann's innovative research on purple bacteria was in the nature of a 'sideline.' For those interested in the details of Engelmann's outstanding career and accomplishments, two biographical accounts are available (Rothschuh 1971, Kamen 1986).
Persistence of the notion that purple bacteria produce oxygen
Studies on the biochemistry of oxygenic photosynthesis
Despite Molisch's experiments and conclusions on the oxygen production question, the assump-
The classic research of Robert Hill (1937) with green plant chloroplasts provided the first con-
56 crete evidence identifying water as the source of the 0 2 produced in oxygenic photosynthesis. Hill (1965) paid tribute to Molisch's earlier experiments as follows; 'In these [Molisch: Bot Zeitg, 62, 1 (1904); Zeit f Bot, 17,577 (1925)], the first successful ones of their type, Molisch found that with non-viable preparations made by grinding dried leaves in water oxygen was produced in light. The oxygen was detected by observation of luminescent bacteria. The activity of the preparations was very small and below the range of the chemical and physical methods of those days. The important conclusion reached by Molisch was that for the production of oxygen in light two components of the system were required: The green insoluble part and the non-green soluble part. Both of these were thermolabile and also, like enzyme systems, sensitive to certain reagents. These experiments indicated that the light and dark processs inferred by F.F. Blackman and by Willst/itter and Stoll from work on living plants might eventually be explained in biochemical terms.'
Molisch in retrospect It is clear that Molisch had a large measure of curiosity about the biological world and an unusually wide span of research interests. Since these traits were coupled with excellent experimental abilities, he left a rich legacy. Among the various significant items in this legacy was the discovery and general characterization of Rhodobacter capsulatus, originally designated as Rhodonostoc capsulatum Molisch. Numerous studies during the 1960s-80s demonstrated that R. capsulatus was metabolically very versatile, and particularly suitable for investigating the fundamental physiological and biochemical properties of purple bacteria. With the discovery of a facile genetic exchange system in R. capsulatus by Marrs (1974), the door was opened to detailed molecular biological analysis of its energy conversion systems and associated processes such as N 2 fixation (Marrs et al. 1977). Determination of the linkage map of the 'photosynthesis gene cluster' in R. capsulatus (Taylor et al. 1983) was the next important milestone, and during the past several years 'Molisch's bacterium' has
clearly emerged as the organism par excellence for further molecular biological analysis of expression and function of photosynthesis genes in purple bacteria [in this connection see: Coleman and Youvan (1990), Sganga and Bauer (1992), Alberti and Hearst (1991)]. Molisch' firm conclusion that the purple bacteria are incapable of producing molecular oxygen should have settled the question in 1907. It is interesting that although Molisch's main interests focused on green plants, he did not feel compelled to 'explain' anoxygenic photosynthesis as a variation of the oxygenic process. Misguided efforts to develop a 'unitary' theory of photosynthetic processes based on 'rationalizing' the absence of oxygen production by the bacteria probably delayed conceptual advances for some time (see Gest 1966, 1982). In connection with the ' 0 2 question,' it is pertinent to note that 0 2 is an effective regulator of photosynthesis in non-sulfur purple bacteria in that in virtually all species, O 2 causes repression of synthesis of the photopigment complex. The elusive mechanism of this phenomenon, first observed by Esmarch (1887) and confirmed by Molisch (1907), is finally being clarified by genetic and molecular biological studies with R. capsulatus, (Sganga and Bauer 1992).
Acknowled gements I am indebted to Prof Alfred Diamant and Gregory Ketcham for aid in the translation of German literature, and to Dr William Ruf for the photograph of the Ingen-Housz starch picture.
References Alberti M and Hearst JE (1991) Sequence of the 46Kb photosynthesis gene cluster of Rhodobacter capsulatus. Photochem Photobiol 53 Supplement: p 122s Brock TD and Madigan MT (1991) Biology of Microorganisms, 6th ed. Prentice Hall, Englewood Cliffs, New Jersey Bulloch W (1938) The Hisotry of Bacteriology. Oxford University Press, Oxford Coleman WJ and Youvan DC (1990) Spectroscopic analysis of genetically modified photosynthetic reaction centers. Ann Rev Biophys Biophys Chem 19:333-367
57 Engelmann Th W (1883) Bacterium photometricum. Ein Beitrag zur vergleichenden Physiologie des Licht- und Farbensinnes. Archiv fiir die gesammte Physiologie des Menschen und der Thiere 30:95-124 Engelmann Th W (1888) 1. Ueber Bacteriopurpurin und seine physiologische Bedeutung. II. Ueber Blutfarbstoff als Mittel zur Untersuchung der Gaswechsels chromophyllhaltiger Pflanzen im Licht und Dunkel. Archiv f/ir die gesammte Physiologie des Menschen und der Thiere 42: 183-188 Esmarch E (1887) Ueber die Reincultur eines Spirillum. Centralbl Bacteriol Parasitenkunde 1:225-230 Fruton JS (1972) Molecules and Life. Wiley-Interscience, New York Gest H (1966) Comparative biochemistry of photosynthetic processes. Nature 209:879-882 Gest H (1982) The comparative biochemistry of photosynthesis: Milestones in a conceptual zigzag. In: Kaplan NO and Robinson A (eds) Cyclotrons to Cytochromes, Essays in Molecular Biology and Chemistry, pp 305-321. Academic Press, New York Gest H (1988) Sunbeams, cucumbers, and purple bacteria. Photosynth Res 19:287-308 Hill R (1937) Oxygen evolved by isolated chloroplasts. Nature: 139:881-882 Hill R (1965) The biochemists' green mansions: The photosynthetic electron-transport chain in plants. In: Campbell PN and Greville GD (eds) Essays in Biochemistry, Vol 1, pp 121-151. Academic Press, London and New York H6fler K (1939a) Hans Molisch. Ber Deut Botan Gesell LVI: 161-199 H6fler K (1939b) Prof Dr Hans Molisch. Rivis Biologia XXVII-Fasc 1-1939-XVII: 3-14 Johnston JA and Brown AH (1954) The effect of light on the oxygen metabolism of the photosynthetic bacterium Rhodospirillum rubrum. Plant Physiol 29:177-182 Kamen MD (1986) On creativity of eye and ear: A commentary on the career of T.W. Engelmann. Proc Amer Phil Soc 130:232-246 Kiister (1937) Hans Molisch. Zeit fiir wiss Mikroskopie 54: 377-378 Marrs BL (1974) Genetic recombination in Rhodopseudomonas capsulata. Proc Natl Acad Sci USA 71:971-973 Marrs B, Wall JD and Gest H (1977) Emergence of the biochemical genetics and molecular biology of photosynthetic bacteria. Trends Biochem Sci 2:105-108 Molisch H (1892) Die Pttanze in ihren Beziehungen zum Eisen. Gustav Fischer, Jena Molisch H (1894) Das Phycoerythrin, seine KrystaUisirbarkeit und chemische Natur. Bot Ztg 52:177-189 Molisch H (1895) Das Phycocyan, ein krystallisirbarer Eiweissk6rper. Bot Ztg 53:131-135 Molisch H (1904) Leuchtende Pflanzen. Gustav Fischer, Jena Molisch H (1905) Ueber amorphes und kristallisierties Anthokyan. Bot Ztg 63:145-162 Molisch H (1906) Zwei neue Purpurbakterien mit Schwebek6rperchen. Bot Ztg 64:223-232 Molisch H (1907) Die Purpurbakterien nach neuen Untersuchungen. Gustav Fischer, Jena Molisch H (1910) Die Eisenbakterien. Gustav Fisher, Jena
Molisch H (1914) Ueber die Herstelhing von Photographien in einem Laubblatte. Sitzungsber Kaiserl Akad Wissen Math-Naturwiss KI CXXIII: 923-931 Molisch H (1920) Populare biologische Vortrfige. Gustav Fischer, Jena Molisch H (1926) Pttanzenbiologie in Japan. Gustav Fischer, Jena Molisch H (1934) Erinnerungen und Welteindriicke eines Naturforschers. Emil Haim, Vienna and Leipzig Reed HS (1949) Jan Ingenhousz, Plant Physiologist, With a History of the Discovery of Photosynthesis. Chronica Botanica, Vol 11, No 5/6. Chronica Botanica Co., Waltham, Mass Rothschuh KE (1971) Theodor Wilhelm Engelmann. In: Gillispie CC (ed) Dictionary of Scientific Biography, Vol IV, pp 371-373. C Scribner's Sons, New York Sganga MW and Bauer CE (1992) Regulatory factors controlling light harvesting and reaction center gene expression in Rhodobacter capsulatus. In press Stephenson M (1949) Bacterial Metabolism, 3rd ed. Longroans Green, London Taylor DP, Cohen SN, Clark WG and Marrs BL (1983) Alignment of genetic and restriction maps of the photosynthesis region of the Rhodopseudomonas capsulata chromosome by conjugation mediated rescue technique. J Bacteriol 154:580-590 van Niel CB (1941) The bacterial photosyntheses and their importance for the general problem of photosynthesis. Advan Enzymol 1:263-328 van Niel CB (1944) The culture, general physiology, morphology, and classification of the non-sulfur purple and brown bacteria. Bacteriol Revs 8:1-118 Walker D (1979) Energy, Plants, and Man. Packard Publishing, Chichester Wheldate M (1916) The Anthocyanin Pigments of Plants. Cambridge University Press
Appendix* On the production of photographs in foliage leaves, by Hans Molisch (From the Plant Physiology Institute, University of Vienna, Second Series, No. 74, presented at the meeting of 15 October 1914) It has been known for some time that starch is formed during carbon dioxide assimilation in the chlorophyll granules of most plants, and that this starch appears only in the parts of the plant exposed to sunlight. If one fastens a strip of black paper on a living, green foliage leaf while it is still on the plant, and exposes it to direct sunlight for a day, cuts the leaf off shortly before sunset and then finally subjects it to Sachs' iodine test, the leaf will tum blue-black only on the previously exposed portions, not however, where it was covered by the black paper because no starch forms in such regions. The * Translation of Sitzungsberichte der Kaiserliche Akademie der Wissenschaften, Mathematische-Naturwissenschaftliche Klasse, Band CXXIII., Abteilung I, pp 923-931, 1914, Wien
58 shape of the paper stands out sharply on such a leaf. This experiment becomes more instructive if, instead of the black paper, one uses a tin stencil in which the letters of a word, for example 'starch', are punched out. The dark letters will then stand out against the light background. I have become convinced that a tin stencil is not at all necessary for this well-known and popular experiment: A normal piece of white paper with distinct black print suffices. I fasten the printed paper on the topside of the leaf, lettering facing upward, with some gum arabic solution applied only at the comers of the paper. The paper must fit tightly. The same procedure is followed as in the previously described experiment with the strip of black paper. The letters stand out sharply after the application of Sachs' iodine test, as the diagram shows (omitted in this translation). Even normal newspaper type is readable under favorable conditions. In some instances the penetrating light causes letters to appear in the leaves of Tropaeolum majus after extraction of the chlorophyll, even before the treatment with iodine. The starch which had formed in the areas of the letters transmitted light differently than the areas which had been shaded, thus making the lettering recognizable. When I saw the lettering of the printed paper stand out so sharply in the leaf after the iodine test, I had the idea that it would perhaps also be possible to produce photographs, or copies of such, in a foliage leaf. From the beginning, .the probability was not exactly great that the fine nuances of light and shadow in a photograph would show up, but as my experiments have demonstrated, I finally had complete success.
The most suitable leaves for such photographic experiments are those which are, as far as possible, fiat, thin, and with little or no hair. The leaf of the Indian cress Tropaeolum majus has proved to be excellent for this purpose. It possesses the aforementioned qualities and also the splendid property that it becomes completely white after extraction of the chlorophyll, making the starch iodine reaction stand out with great clarity.
Sketch of the experimental arrangement, r, Frame i n Which the negative is inserted. This lies on the leaf b. Between leaf and negative is the coverglass d, to protect the gelatin layer. The upper side of the leaf is gently pressed on the negative n by the wood lattice h and the two wooden clasps s and s'.
In order to produce a photograph in a leaf, one proceeds as follows. Select an unblemished leaf on a healthy, growing plant and place a contrast-rich negative of a photograph with its image facing upward on the upper side of the leaf. Because of transpiration, the leaf will sweat; dewdrops will form which can liquefy, tear and damage the gelatin of the negative. This can easily be avoided by covering the negative, or at least the part to be 'photographed' with a very thin cover slip of corresponding size and fastening it with a trace of gum arabic. Thus, a cover slip lies between the upper side of the leaf and the negative, and serves as a protective cover for the gelatin layer. It is essential for the success of the experiment and for the sharpness of the photographic image that the negative fit closely to the leaf without, however, pressing or damaging it. I accomplished this by using a lattice of thin wooden sticks pressed lightly and softly against the underside of the leaf with two weakly sprung clasps. The negative itself lies in a copying flame and this in turn is held in the proper position by a support. A sketch of the experimental arrangement is shown in the text figure. The experiment is performed,on a clear, sunny day and it is best to allow direct sunlight to illuminate the leaf, covered with the negative, from approximately sunrise to sunset. In the evening, the leaf is cut off and immediately immersed in boiling water for thirty seconds to a minute in order to kill the leaf and thicken the starch. The leaf is then freed of chlorophyll with warm alcohol, and the now blanched leaf again immersed in boiling water for a half minute. Finally, it is placed in a tincture of iodine solution, of a beer-brown color, which has been diluted with water and weakly acidified with hydrochloric or acetic acid. If the experiment is successful, the positive of the negative used will appear after a short time with a sharpness that makes it possible to immediately recognize the photograph of a particular person (see Figs. 2-4 of the p l a t e . . , omitted in this
translation). This experiment provides convincing evidence of the extraordinary detail that can be evoked by light rays and the accuracy with which they 'produce' starch according to their intensity. In this way, the highlights and shadows of a photograph, in their sudden or gradual transitions, are reproduced through the color tones of the starch iodine reaction. This is all the more astonishing since the numerous arteries crisscrossing the leaf, the thousands of cell walls and the various contents of the cells must invariably distort the clarity of the picture. In a comparison of the photographic plate with the foliage leaf, the chlorophyll apparatus corresponds to the lightsensitive silver salt, the starch granule to the silver granule, and the starch iodine reaction to the developer. Although the formation of starch in the light does not occur with the lightning-like rapidity of reduction of the silver salt, but rather requires a relatively long time, both processes lead to the same result, the production of a photographic image. Strictly speaking, one should only call this a copying procedure, since in my experiment I did not produce a photograph, but only a copy of one. But there can be no doubt that according to my procedure any given object could be photographed. One would only have to expose the leaf, in place of a photographic plate, to light in a chamber. The effect must
59 in essence be the same, only one would first obtain a negative in the leaf. I chose the easier way and directly used the negative as the object for the 'chlorophyll plate' because I was only interested in showing that the refinements of a photographic image appear in the leaf. It struck me that in the development of a picture in the leaf, light stripes appeared in the dark starch iodine portions, and corresponded to the wooden sticks of the lattice used to press the leaf against the negative. Carbon dioxide assimilation, and thus starch formation, does not occur beneath the wooden sticks because access of carbon dioxide from the air to the stomates is significantly impeded. Thus, the light stripes. Therefore, in order not to diminish the sharpness of the picture, it appears to be a good idea to choose thin, round sticks for the lattice and not use too many of them. I was well served by round wooden sticks of approximately 1 mm diameter, which were spaced ½cm apart in the lattice. The fact that the formation of starch is already impeded or even fully blocked beneath such wooden sticks is of great interest. It clearly illustrates that provision of carbon dioxide from the near vicinity to the places covered by the wooden sticks is very deficient; in other words, the carbon dioxide which, between the sticks, flows through the stomates into the leaf is at once captured and processed by the chlorophyll apparatus so that none or very little of it proceeds by diffusion to the chlorophyll granules situated beneath the wooden sticks 1. Stahl 2 has shown that coating the underside of the leaf with liquified cocoa butter wax completely prevents the assimilation of carbon dioxide as a result of blockage of the stomates. And from my observations it is clear that simply placing a wooden stick on the stomates impedes access of carbon dioxide and prevents its assimilation under the stick. The air must apparently be kept in motion over the stomates through diffusion and air currents so that 'new' carbon dioxide can constantly flow in. The air stagnates beneath the wooden sticks, and thus too little carbon dioxide reaches these positions and assimilation is hindered. If my explanation is correct, then a closely fitting cover slip attached to the underside of the leaf should also depress assimilation. This is truly the case, as I have determined many times. Such a l e a f - I worked mostly with Tropaeolum - shows a distinct picture of the cover slip after Sachs' iodine test is performed; its shape is revealed by a noticeably lighter color. Only healthy, flourishing plants should be selected for such photographic experiments because in neglected, poorly tended plants the formation of starch is retarded and starch utilization at night is deficient. It is therefore not advisable to
1 See Zijlstra, Carbon dioxide transport in leaves, Groningen (1909). 2 Stahl E, Researches on transpiration and assimilation, Bot Ztg, 1894, p 129.
use potted plants. I cultivate my plants in large, flat crates or experiment with plants flourishing in the open. In the months of June and July, such plants (Tropaeolum) utilize the starch of their leaves fully during the night, and one can be almost certain of starting the experiment in early morning with leaves free of starch. A starch-free leaf is an essential requirement for the success of the experiment, just as an unexposed photographic plate is required for every ordinary photograph. If one has produced a successful photograph in a leaf, one wishes to preserve it. This can be achieved in one of two ways, by 'dry' or 'wet' methods: 1. Immediately after the iodine treatment, place the leaf in a wide-mouthed bottle which can be sealed tightly and which is filled with a saturated iodine/water solution. Should some of the iodine vaporize after a while, the solution can easily be brought to a saturated state by adding an iodine crystal. 2. Immediately after the iodine treatment and while it is still wet, take the leaf, minus the stem, and spread it out on a glass plate (such as are used for photographic plates), avoiding air bubbles, with the top side up. Then allow the water to evaporate at room temperature and cover the dry leaf, which normally adheres firmly to the plate, with a second glass plate. Cover both plates at the edges with black paper, as is common for the mounting of transparencies. Enclosed in this way, the 'photograph' remains preserved. Even after a year, I have perceived no change in such photographs; they lost none of their sharpness. In these experiments I encountered another striking phenomenon which should be noted here briefly. Leaves were submerged for a few minutes in boiling water, removed, and immediately placed securely, top or bottom side, on a wellpolished glass p l a t e . . , best of all on a mirrored surface. When removed after the water had evaporated, the contact side appeared strikingly shiny, as if laquered. Detaching the leaf from the plate will be most successful if one goes around the edge of the leaf with a thin knife and then lifts it off. Should difficulties occur during detachment, it is usually an indication that the glass plate was not sufficiently polished before adhering the leaf to it. The shiny, reflective surface stands out especially nicely in leaves (Tropaeolum, Amicia, etc.) which were, after the Sachs iodine treatment, allowed to dry on a glass plate and then lifted off in the manner described. During submersion of the leaf in boiling water the leaf's surface acquires a mucilaginous character and this fine mucous coating, which forms a continuous layer on the surface of the leaf, in all probability causes the unusual shine.
